In autoimmune and/or inflammatory disorders, the immune system triggers an inflammatory response when there are no foreign substances to fight and the body's normally protective immune system cause damage to its own tissues by mistakenly attacking self. There are many different autoimmune disorders which affect the body in different ways. For example, the brain is affected in individuals with multiple sclerosis, the gut is affected in individuals with Crohn's disease, and the synovium, bone and cartilage of various joints are affected in individuals with rheumatoid arthritis.
B-cell malignancies constitute an important group of cancer that include B-cell non-Hodgkin's lymphoma (NHL), B-cell chronic lymphocytic leukaemia (B-CLL) and hairy cell leukaemia and B-cell acute lymphocytic leukaemia (B-ALL).
Currently, cancer therapy may involve surgery, chemotherapy, hormonal therapy and/or radiation treatment to eradicate neoplastic cells in a patient. Recently, cancer therapy could also involve biological therapy or immunotherapy.
There is a significant need for alternative cancer treatments, particularly for treatment of cancer that has proved refractory to standard cancer treatments, such as surgery, radiation therapy, chemotherapy, and hormonal therapy.
A promising alternative is immunotherapy, in which cancer cells are specifically targeted by cancer antigen-specific antibodies.
Major efforts have been directed at harnessing the specificity of the immune response, for example, hybridoma technology has enabled the development of tumor selective monoclonal antibodies and in the past few years, the Food and Drug Administration has approved the first MAbs for cancer therapy: Rituxan™ (anti-CD20) for non-Hodgkin's Lymphoma and Herceptin™ [anti-(c-erb-2/HER-2)] for metastatic breast cancer.
Rituxan™ (common name is rituximab) is a chimeric mouse-human monoclonal antibody to human CD20, a 35 kilo Daltons, four transmembrane-spanning protein found on the surface of the majority of B-cells in peripheral blood and lymphoid tissue.
The antibody therapy (Rituxan™, U.S. Pat. No. 5,736,137) was approved by the United States Food and Drug Administration (FDA) for the treatment of relapsed or refractory low grade or follicular, CD20-positive B-cell non-Hodgkin's lymphoma.
In addition, lymphoma therapies employing radiolabeled anti-CD20 antibodies have been described in U.S. Pat. Nos. 5,595,721, 5,843,398, 6,015,542, and 6,090,365.
In oncology, Rituxan™/MabThera™ is also indicated in the US for the treatment of relapsed or refractory, low-grade or follicular, CD20-positive, B-cell NHL as a single agent, for the treatment of NHL for previously untreated follicular, CD20-positive, B-cell NHL in combination with cyclophosphamide, vincristine, prednisolone (CVP) chemotherapy for non-progressing (including stable disease), low-grade, CD20 positive, B-cell NHL as a single agent, after first-line CVP chemotherapy and for previously untreated diffuse large B-cell, CD20-positive, NHL in combination with standard chemotherapy (CHOP) or other anthracycline-based chemotherapy regimens.
In oncology, Rituxan™/MabThera™ is indicated in the EU for the treatment of patients with previously untreated or relapsed/refractory chronic lymphocytic leukemia (CLL) in combination with chemotherapy, for the treatment of previously untreated patients with stage III-IV follicular lymphoma in combination with chemotherapy, as maintenance therapy for patients with relapsed/refractory follicular lymphoma responding to induction therapy with chemotherapy with or without Rituxan™/MabThera™, for the treatment of patients with CD20-positive diffuse large B cell non-Hodgkin's lymphoma (NHL) in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, prednisolone) chemotherapy and as monotherapy for treatment of patients with stage III-IV follicular lymphoma who are chemo resistant or are in their second or subsequent relapse after chemotherapy.
In addition, in rheumatology Rituxan™/MabThera™ in combination with methotrexate is indicated for the treatment of adult patients with severe active rheumatoid arthritis who have had an inadequate response or intolerance to other disease-modifying anti-rheumatic drugs (DMARD) including one or more tumor necrosis factor (TNF) inhibitor therapies. MabThera is known as Rituxan™ in the United States, Japan and Canada.
Alemtuzumab is another antibody targeting CD52 that is approved for use in relapsed chronic lymphocytic leukemia (CLL) but is associated with significant toxicity because of the ubiquitous expression of the target antigen on most normal immune cells including T-cells and natural killer (NK) cells.
However, some resistance to rituximab treatment has appeared. Resistance may appear through a variety of mechanisms, some of which lead to loss CD20 expression and resistance to further rituximab treatment. Resistance may appear which may lead to loss or modulation of CD20 expression and is characterized by the lack of efficacy of repeated rituximab treatment or in some cases by the lack of efficacy of primary rituximab treatment. Resistance also occurs in lymphoma cases constitutively lacking CD20 expression, including some B-cell lymphomas such as plasmablastic lymphomas. Moreover, CD20+ patients may not respond to, or acquire resistance to Rituxan® therapy.
Also, under conditions of high B-cell burden, exhaustion of the body's effector mechanisms, for example, NK-cell-mediated killing, may lead to substantial decreases in the immunotherapeutic efficacy of this MAb. Moreover, rituximab treatment of patients with chronic lymphocytic leukemia and high levels of circulating B-cells can lead to removal of CD20 from the cells, thus allowing them to persist and resist clearance.
On the basis of the success and limitations of rituximab and alemtuzumab, in particular resistance and relapse phenomena with rituximab therapy, identification of alternatives antibodies targeting alternative antigens on B-cells is needed.
CD19 is a 95-kDa glycoprotein member of the immunoglobulin (Ig) super family. CD19 is expressed on follicular dendritic cells and all B-cells from their early pre-B-cell stage until the time of plasma cell differentiation. CD19 surface expression is tightly regulated during B-cell development with higher levels seen in more mature cells and CD5+ (B-1) B-cells. CD19 is expressed later than CD20 through the plasmablast stage of B-cell differentiation. Consequently, CD19 expression is relatively high in many pre-B and immature B-lymphoblastic leukemias and B-cell malignancies in which CD20 is poorly expressed. CD19+ plasmablasts may also play a role in the perpetuation of autoimmune diseases.
CD19 is expressed on the surface of B-cells as a multiple molecular complex with CD21, CD81 and CD225. Together with this complex, CD19 is involved in co-signalling with the B-cell receptor and plays a role in the control of differentiation, activation and proliferation of B-lymphoid cells (Sato et al., 1997).
CD19 is present on the blasts of different types of human B-cell malignancies including pro- and pre-B-cell acute lymphoblastic leukaemia (ALL), common ALL (cALL) of children and young adults, NHL, B-CLL and hairy-cell leukaemia (HCL). It is not shed from malignant cells and is internalized after binding of some antibodies (Press et al., 1989). Antigen density ranges from 10 000 to 30 000 molecules per cell on healthy peripheral B-cells, and from 7 000 to 30 000 molecules per cell on malignant cells from a variety of lymphoid cancers (Olejniczak et al., 1983).
CD19 is expressed more broadly and earlier in B-cell development than CD20, which is targeted by Rituxan® and so could have applications in a wider range of cancers including non-Hodgkin's lymphoma and acute lymphoblastic leukaemia as well as CLL.
CD19 has been a focus of immunotherapy development for over 20 years, but initial clinical trials with monoclonal antibodies to CD19 did not result in durable effects despite demonstrating responses in some patients either as a single agent or in combination with other therapeutic agents (Hekman et al., 1991; Vlasved et al., 1995).
Several CD19-specific antibodies have been evaluated for the treatment of B-lineage malignancies in vitro, in mouse models, and in clinical trials. These have included unmodified anti-CD19 antibodies, antibody-drug conjugates, and bispecific antibodies targeting CD19 and CD3 or CD16 to engage cytotoxic lymphocyte effector functions.
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations or alternative post-translational modifications that may be present in minor amounts, whether produced from hydridomas or recombinant DNA techniques.
Antibodies are proteins, which exhibit binding specificity to a specific antigen. Native antibodies are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly intrachain disulfide bridges.
Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
The term ‘variable’ refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are responsible for the binding specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed through the variable domains of antibodies. It is concentrated in three segments called complementarily determining regions (CDRs) both in the light chain and the heavy chain variable domains.
The more highly conserved portions of the variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (Kabat et al., 1991).
The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgG, IgD, IgE and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgG1, IgG2, IgG3 and IgG4; IgA1 and IgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. Of the various human immunoglobulin classes, only IgG1, IgG2, IgG3 and IgM are known to activate complement.
Immune effector functions which have been shown to contribute to antibody-mediated cytotoxicity include antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).
Cytotoxicity may also be mediated via anti-proliferative effects. The mechanism of antibody modulation of tumor cell proliferation is poorly understood. However, advances in understanding the interactions of antibodies with Fcγ receptors (FcgR) on immune effector cells have allowed the engineering of antibodies with significantly improved effector function.
The mechanism of action of MAbs is complex and appears to vary for different MAbs. There are multiple mechanisms by which MAbs cause target cell death. These include apoptosis, CDC, ADCC and inhibition of signal transduction.
The most studied is ADCC, which is mediated by natural killer (NK) cells. This involves binding of the Fab portion of an antibody to a specific epitope on a cancer cell and subsequent binding of the Fc portion of the antibody to the Fc receptor on the NK cells. This triggers release of perforin, and granzyme that leads to DNA degradation, induces apoptosis and results in cell death. Among the different receptors for the Fc portion of MAbs, the FcγRIIIa plays a major role in ADCC.
Previous research has shown that a polymorphism of the FcγRIIIa gene encodes for either a phenylalanine (F) or a valine (V) at amino acid 158. Expression of the valine isoform correlates with increased affinity and binding to MAbs (Rowland et al., 1993; Sapra et al., 2002; Molhoj et al., 2007). Some clinical studies have supported this finding, with greater clinical response to rituximab in patients with non-Hodgkin's lymphoma who display the V/V polymorphism (Cartron et al., 2002, Bruenke et al. 2005, Bargou et al., 2008).
WO1999051642 describes a variant human IgG Fc region comprising an amino acid substitution at positions 270 or 329, or at two or more of positions 270, 322, 329, and 331. These modifications aim at increasing the CDC and ADCC effector functions.
WO2002080987 describes a method for treating a B-cell malignancy in a subject comprising administering an anti-CD19 immunotoxin, more particularly a humanized or human, monoclonal antibody. The B-cell malignancy may be one which comprises B-cells that do not express CD20.
WO2007024249 is related to the modification of a human IgG Fc region in order to confer an increase effector cell function mediated by FcγR, especially ADCC. The Fc region comprises specific modifications at various amino acid positions.
WO2008022152 is also related to antibodies that target CD19, wherein the antibodies comprise modifications to Fc receptors and alter the ability of the antibodies to mediate one or more effector functions, including ADCC, ADCP and CDC. ADCC assays are illustrated.
Other patent applications related to anti-CD19 antibodies include WO2009052431, WO2009054863, WO2008031056, WO2007076950, WO2007082715, WO2004106381, WO1996036360, WO1991013974, U.S. Pat. No. 7,462,352, US20070166306 and WO2005092925.
US20090098124 also relates to engineering of antibodies with variant heavy chains containing the Fc region of IgG2, 3 or 4, having one or more amino acid modifications. A number of mutations and group of mutations within the Fc region are proposed. One of them is substitution at position 243 with leucine, at position 292 with proline, at position 300 with leucine, at position 305 with isoleucine, and at position 396 with leucine (MgFc88).
J. B. Stavenhagen et al. (Cancer Res. 2007, 67 (18): 8882-8890) disclose Fc optimization of therapeutic antibodies. The highest levels of ADCC were obtained with mutant 18 (F243L/R292P/Y300L/V305I/P396L). Anti-CD20 and anti-CD32B monoclonal antibodies having this mutated Fc are disclosed.
Despite CD19 is a B-cell specific antigen expressed on chronic lymphocytic leukemia (CLL) cells, to date CD19 has not been effectively targeted with therapeutic monoclonal antibodies. The authors describe XmAb5574, a novel engineered anti-CD19 monoclonal antibody produced by human 293E cells with a modified Fc domain designed to enhance binding of FcγRIIIa. They demonstrate that this antibody mediates potent ADCC, modest direct cytotoxicity and ADCP, but no CDC (Awan et al., 2010).
Moreover, glycoproteins mediate many essential functions in human beings including catalysis, signalling, cell-cell communication and molecular recognition and association. Many glycoproteins have been exploited for therapeutic purposes. The oligosaccharide component of protein can affect properties relevant to the efficacy of a therapeutic glycoprotein, including physical stability, resistance to protease attack, interactions with the immune system, pharmacokinetics, and specific biological activity. Such properties may depend not only on the presence or absence, but also on the specific structures, of oligosaccharides. For example, certain oligosaccharide structures mediate rapid clearance of the glycoprotein from the bloodstream through interactions, with specific carbohydrate binding proteins, while others can be bound by antibodies and trigger undesired immune reactions (Jenkins et al., 1996).
Most of the existing therapeutic antibodies that have been licensed and developed as medical agents are of the human IgG1 isotype, the molecular weight of which is ˜150 kDa. Human IgG1 is a glycoprotein bearing two N-linked biantennary complex-type oligosaccharides bound to the antibody constant region (Fc), in which the majority of the oligosaccharides are core fucosylated, and it exercises the effector functions of ADCC and CDC through the interaction of the Fc with either leukocyte receptors (FcγRs) or complement.
Recently, therapeutic antibodies have been shown to improve overall survival as well as time to disease progression in a variety of human malignancies, such as breast, colon and haematological cancers, and genetic analysis of FcγR polymorphisms of cancer patients has demonstrated that ADCC is a major antineoplasm mechanism responsible for clinical efficacy. However, the ADCC of existing licensed therapeutic antibodies has been found to be strongly inhibited by serum due to non-specific IgG competing for binding of the therapeutics to FcγRIIIa on natural killer cells, which leads to the requirement of a significant amount of drug and very high costs associated with such therapies.
The enhanced ADCC of non-fucosylated forms of therapeutic antibodies through improved FcγRIIIa binding is shown to be inhibited by the fucosylated counterparts. In fact, non-fucosylated therapeutic antibodies, not including the fucosylated forms, exhibit the strongest and most saturable in vitro and ex vivo ADCC among such antibody variants with improved FcγRIIIa binding as those bearing naturally occurring oligosaccharide heterogeneities and artificial amino acid mutations, even in the presence of plasma IgG.
Inhibiting glycosylation of a human IgG1, by culturing in the presence of tunicamycin, causes, for example, a 50-fold decrease in the affinity of this antibody for the FcγRI receptor present on monocytes and macrophages (Leatherbarrow et al., 1990). Binding to the FcγRIII receptor is also affected by the loss of carbohydrates on IgG, since it has been described that a non-glycosylated IgG3 is incapable of inducing lysis of the ADCC type via the FcγRIII receptor of NK cells (Lund et al., 1995). However, beyond the necessary presence of the glycan-containing residues, it is more precisely the heterogeneity of their structure which may result in differences in the ability to initiate effector functions.
Studies have been conducted to investigate the function of oligosaccharide residue on antibody biological activities. It has been shown that sialic acid of IgG has no effect on ADCC (Boyd et al., 1995). Several reports have shown that Gal residues enhance ADCC (Kumpel et al., 1994). Bisecting GlcNac, which is a beta,4-GlcNac residue transferred to a core beta-mannose (Man) residue, has been implicated in biological residue of therapeutic antibodies (Lifely et al., 1995,) have revealed the effect of fucosylated oligosaccharide on antibody effector functions; the Fuc-deficient IgG1 have shown 50-fold increased binding to FcγRIII and enhanced ADCC.
Today, a wide range of recombinant proteins for therapeutic applications (i.e cancer, inflammatory diseases . . . ) are composed of glycosylated monoclonal antibodies. For therapeutic and economical reasons, there is a large interest in obtaining higher specific antibody activity. One way to obtain large increases in potency, while maintaining a simple production process in cell line and potentially avoiding significant, undesirable side effects, is to enhance the natural, cell-mediated effector functions of MAbs. Consequently, engineering the oligosaccharides of IgGs may yield optimized ADCC which is considered to be a major function of some of the therapeutic antibodies, although antibodies have multiple therapeutic functions (e.g. antigen binding, induction of apoptosis, and CDC).
In general, chimeric and humanized antibodies are prepared using genetic recombination techniques and produced using CHO cells as the host cell. In order to modify the sugar chain structure of the antibodies, various methods have been attempted, say application of an inhibitor against an enzyme relating to the modification of a sugar chain, selection of a CHO cell mutant, or introduction of a gene encoding an enzyme relating to the modification of a sugar chain.
GLYCART BIOTECHNOLOGY AG (Zurich, CH) has expressed N-acetyl-glucosaminyltransferase III (GnTIII) which catalyzes the addition of the bisecting GlcNac residue to the N-linked oligosaccharide, in a Chinese hamster ovary (CHO) cell line, and showed a greater ADCC of IgG1 antibody produced (WO 99/54342; WO 03/011878; WO 2005/044859).
By removing or supplanting fucose from the Fc portion of the antibody, KYOWA HAKKO KOGYO (Tokyo, Japan) has enhanced Fc binding and improved ADCC, and thus the efficacy of the MAb (U.S. Pat. No. 6,946,292).
More recently, Laboratoire Français du Fractionnement et des Biotechnologies (LFB) (France) showed that the ratio Fuc/Gal in MAb oligosaccharide should be equal or lower than 0.6 to get antibodies with a high ADCC (FR 2 861 080).
P. M. Cardarelli et al. (Cancer Immunol. Immunother. 2010, 59:257-265) produce an anti-CD19 antibody in Ms-704PF CHO cells deficient in the FUT8 gene which encodes alpha1,6-fucosyltransferase. Non-fucosylation of the antibody in this paper requires the engineering of an enzymes-deficient cell line. This paper does consider amino acid mutations.
Effector functions such as CDC and ADCC are effector functions that may be important for the clinical efficacy of MAbs. All of these effector functions are mediated by the antibody Fc region and let authors to attempt amino acid modifications with more or less success. Glycosylation, especially fucosylation of the Fc region have a dramatic influence on the efficacy of an antibody. This let the authors to modify the conditions of production of the antibodies in the CHO cells in order to change the glycosylation profile in an attempt here again to improve some effector functions, with more or less success one again.
Complement activation is acknowledged to be a major factor in several systemic inflammatory disorders and autoimmune diseases such as reperfusion injury, rheumatoid arthritis and Guillain Barré Syndrome (Kirschfink et al., 2001). There is also a precedent for involvement of complement in local inflammation generated by antibody-antigen reactions and pain. The complement system consists of serum and membrane bound proteins that interact with each other and with immune cell molecules in a stylized cascade and serves to amplify the original signal. Many complement proteins act as receptor-specific activator molecules.
The role of complement in rituximab therapy remains controversial. Although complement is consumed during rituximab treatment (Van der Kolk et al., 2001) and on some occasions cells remaining or emerging after treatment appear to have been selected to express increased levels of complement defence molecules (Bannerji et al., 2003; Treon et al., 2001). It has also been shown that the expression of complement defense molecules on tumor cells does not predict clinical outcome, and no correlation exists between in vitro complement sensitivity and subsequent therapeutic response (Weng et al., 2001).
Moreover, recent findings indicate that complement activation may indeed be detrimental to the therapeutic activity of rituximab. First, the C3b deposition after rituximab engagement of CLL cells is thought to promote loss of CD20 and thereby therapeutic efficacy through shaving reaction (Li et al., 2007). Second, it has been suggested that deposited C3b may inhibit the interaction between the Fc region of rituximab and CD16 on NK cells, thereby limiting its ability to induce NK activation (Wang et al., 2008).
Thus, complement (inactivated C3b), rather than promoting tumor destruction, could actually impair the therapeutic efficacy of rituximab or of others therapeutic MAbs by promoting loss of CD20 or of others targets and blocking ADCC.
Several side effects encountered during rituximab infusion, including serious ones even leading to death, may eventually be related to CDC activation (Bienvenu 2001, Winkler 1999, Van der Kolk 2001,)
In this context, to facilitate clinical development of the chimeric MAb anti-CD19 in some clinical indication, the inventors selected mutations inducing no complement activation with maximal induction of ADCC.
A method of enhancing the ADCC of the chimeric MAb anti-CD19 MAb 4G7 was disclosed in US 2007/0166306. The MAb 4G7 was produced by using the human mammalian 293T cell line in the presence of a beta (1,4)-N-acetylglucosaminyltransferase III (GnTIII) enzyme, under conditions effective to produce in the antibody, an Fc fragment characterized by Asn297-linked oligosaccharides containing (1) at least 60% N-acetylglucosamine biselecting oligosaccharides and (2) only 10% of non fucosylated N-acetylglucosamine biselecting oligosaccharides.
H. M. Horton et al. (Cancer Res. 2008, 68 (19): 8049-8057) describe an Fc-engineered anti-CD19 antibody, having S239D and 1332E mutations. This Fc domain called herein Fc14 was compared to the Fc domain of the invention called chR005-Fc20 (F243L/R292P/Y300L/V305L/P396L). The Fc domain of the invention displays ADCC in the presence of whole blood effector cells whereas no significant ADCC activity was detected with Fc14 in the same conditions in the presence of whole blood containing circulating natural immunoglobulins.
Mammalian cells are the preferred hosts for production of therapeutic glycoproteins, due to their capability to glycosylate proteins in the most compatible form for human applications (Jenkis et al., 1996). Bacteria very rarely glycosylates proteins, and like other type of common hosts, such as yeasts, filamentous fungi, insect and plant cells yield glycosylation patterns associated with rapid clearance from the blood stream.
Among mammalian cells, Chinese hamster ovary (CHO) cells have been most commonly used during the last two decades. In addition to giving suitable glycosylation patterns, these cells allow consistent generation of genetically stable, highly productive clonal cell lines. They can be cultured to high densities in simple bioreactors using serum-free media, and permit the development of safe and reproducible bioprocesses. Other commonly used animal cells include baby hamster kidney (BHK) cells, NSO- and SP2/0-mouse myeloma cells. Production from transgenic animals has also been tested (Jenkins et al., 1996).
Since the sugar chain structure plays a remarkably important role in the effector function of antibodies and differences are observed in the sugar chain structure of glycoproteins expressed by host cells, development of a host cell which can be used for the production of an antibody having higher effector function has been an objective.
In order to modify the sugar chain structure of the produced glycoprotein, various methods have been attempted, such as (1) application of an inhibitor against an enzyme relating to the modification of a sugar chain, (2) selection of a cell mutant, (3) introduction of a gene encoding an enzyme relating to the modification of a sugar chain, and the like. Specific examples are described below.
Examples of an inhibitor against an enzyme relating to the modification of a sugar chain includes tunicamycin which selectively inhibits formation of GlcNAc-P-P-Dol which is the first step of the formation of a core oligosaccharide which is a precursor of an N-glycoside-linked sugar chain, castanospermin and W-methyl-1-deoxynojirimycin which are inhibitors of glycosidase I, bromocondulitol which is an inhibitor of glycosidase II, 1-deoxynojirimycin and 1,4-dioxy-1,4-imino-D-mannitol which are inhibitors of mannosidase I, swainsonine which is an inhibitor of mannosidase II, swainsonine which is an inhibitor of mannosidase II and the like. Examples of an inhibitor specific for a glycosyltransferase include deoxy derivatives of substrates against N-acetylglucosamine transferase V (GnTV) and the like. Also it is known that 1-deoxynojirimycin inhibits synthesis of a complex type sugar chain and increases the ratio of high mannose type and hybrid type sugar chains (Glycobiology series 2—Destiny of Sugar Chain in Cell, edited by Katsutaka Nagai, Senichiro Hakomori and Akira Kobata, 1993).
Cell mutants regarding the activity of an enzyme relating to the modification of a sugar chain are mainly selected and obtained as a lectin-resistant cell line. For example, CHO cell mutants having various sugar chain structures have been obtained as a lectin-resistant cell line using a lectin such as WGA (wheat-germ agglutinin derived from T. vulgaris), ConA (cocanavalin A derived from C. ensiformis), RIC (a toxin derived from R. communis), L-PHA (leucoagglutinin derived from P. vulgaris), LCA (lentil agglutinin derived from L. culinaris), PSA (pea lectin derived from P. sativum) or the like (Genet et al., 1986).
As an example of the modification of the sugar chain structure of a product obtained by introducing the gene of an enzyme relating to the modification of a sugar chain, into a host cell, it has been reported that a protein in which a number of sialic acid is added to the non-reducing end of the sugar chain can be produced by introducing rat β-galactosidase-a-5,6-sialyltransferase into CHO cell. Different types of glycoprotein-modifying glycosyl transferase may be also expressed in the host system such as GnT III, or, alternatively, b (1,4)-N-acetylglucosaminyltransferase V (GnT V), β(1,4)-galactosyl transferase (GalT) and mannosidase II (Man II).
WO20070166306 is related to the use of avian embryonic derived stem cell lines, named EBx®, for the production of proteins and more specifically glycoproteins such as antibodies that are less fucosylated than with usual CHO cells.
Ramesh Jassal et al. generate sialylation of anti NIP IgG3 antibody with FA243 mutation or by using a rat α2,6-sialyltransferase transfected CHO-K1 cell line. The FA243 IgG3 having both α2,6 and α2,3 silaylation restored target cell lysis by complement.
John Lund et al. (Journal of Immunology, 1996, vol. 157, no. 11, pp 4963-4969) describe that aglycosylated human chimeric IgG3 retained a significant capacity to bind human C1q and trigger lysis mediated through guinea pig C.
The present inventors have evaluated the glycosylation profile of various Fc chimeric variant antibodies of human IgG1 subclass directed against the CD19 antigen produced by the Chinese hamster ovary cells CHO DG44 cell, (purchased by ECACC) unmodified and untreated with glycosylation inhibitors. By analyzing and comparing structures of the sugar chains of the chR005-1 Fc0 and the optimized chR005-1 Fc20 variant antibodies produced, the present invention provides that a wild-type CHO host cell without any engineering expresses an interesting and valuable glycosylation profile, especially a low fucose level and/or a high oligomannose level and/or presence of sialic acid following Fc20 amino acid mutation.